OPINION of the French Agency for Food, Environmental and Occupational Health & Safety

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ANSES Request No SA-330 Maisons-Alfort, 7 April 2011 The Director General OPINION of the French Agency for Food, Environmental and Occupational Health & Safety on Methods of detection and enumeration
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ANSES Request No SA-330 Maisons-Alfort, 7 April 2011 The Director General OPINION of the French Agency for Food, Environmental and Occupational Health & Safety on Methods of detection and enumeration of Legionella in water ANSES s public health mission involves ensuring environmental, occupational and food safety as well as assessing the potential health risks they may entail. It provides the competent authorities with the necessary information concerning these risks as well as the requisite expertise and technical support for drafting legislative and statutory provisions and implementing risk management strategies (Article L of the French Public Health Code). 1. OVERVIEW OF THE QUESTION On 29 July 2009 the Directorate General for Health (DGS) and the Directorate General for Risk Prevention (DGPR) asked the French Agency for Environmental and Occupational Health Safety (AFSSET) 1 to: 1. Describe the known pre-analysis (preparation of the sample for analysis) and analysis methods developed specifically for the detection and enumeration of Legionella in water (culture, molecular biology [Polymerase Chain Reaction (PCR)], immunodetection, etc.). The particular purpose of this description was to identify the issues that these methods and their theoretical limits address. It was to be based on a review of the literature; 2. Study the suitability (advantages and disadvantages) of implementing these analytical methods for monitoring the safety of hot water and regulatory supervision of the water supply system for cooling towers, according to their respective requirements and constraints. This study sought first to examine the advantages and disadvantages of the different methods, especially with respect to technical feasibility and cost constraints; Second, if the methods were shown to contribute added technical value compared with the culture-based method, the study should assess whether it would be appropriate to use them within the framework of the regulatory controls. The regulatory thresholds for the methods identified could be discussed and suggestions made for their revision, as required. 3. If necessary, identify the experimental assays with which to validate or invalidate the hypotheses put forward on the suitability of the methods listed, and where appropriate, initiate the required studies. 1 On 1 July 2010, AFSSET and the French Food Safety Agency (AFSSA) merged and became the French Agency for Food, Environmental and Occupational Health & Safety (ANSES). 1 / 11 ANSES Request No SA BACKGROUND The incidence of legionellosis has decreased in France since 2006, stabilising at around 1200 reported cases per year (1206 cases were recorded in 2009, Weekly Epidemiological Bulletin [Bulletin épidémiologique hebdomadaire] [WEB-BEH] No of 27 July 2010), largely due to improved identification of potential sources of contamination. Environmental monitoring of Legionella spp. or of Legionella pneumophila is regulated: it is compulsory: o for mineral waters intended for therapeutic uses in spas, once a month at the most sensitive points of use (Ministerial Order of 19 June 2000) 2 ; o in cooling towers, once a month (if authorised) or every two months (if reported) during the facility s operating period (Ministerial Orders of 13 December 2004) 3 ; o in the hot water systems of health-care facilities, health and social services centres for housing the elderly, other health and social services centres, hotels, tourist accommodations, campsites and correctional facilities, once a year at representative points of use (Ministerial Order of 1 February 2010) 4 ; effective 1 January 2012, the Ministerial Order of 1 February 2010 makes it compulsory in other buildings open to the public, once a year, at representative points of use in the hot water network. This regulation requires the use of the culture-based method, as described in the French Standard NF T Recherche et dénombrement de Legionella spp. et de Legionella pneumophila par culture sur milieux gélosés (Screening and enumeration of Legionella spp. and Legionella pneumophila by culture on agar media). In the request letter, the DGS and the DGPR emphasise that the culture method, as described in the above-referenced French Standard, has certain disadvantages: the return of final results requires a minimum period of eight days after culturing (however, interim results can be produced within three to five days). This time limit may present a challenge for the management of the facilities concerned, especially for lifting restrictive water use measures; this method does not take into account all the various forms of Legionella that may be found in water. Indeed, it only enables counting the free, viable and culturable Legionella in water samples. 3. ORGANISATION OF THE EXPERT APPRAISAL This expert appraisal was carried out in accordance with the French Standard NF X Quality in Expert Appraisal Activities General competency requirements for an expert appraisal (May 2003) to ensure compliance with the following points: competence, independence and transparency, while at the same time ensuring traceability. These issues fall within the area of expertise of the Expert Committee (CES) for Assessment of risks linked to water and biological agents. ANSES entrusted the expert appraisal to the Working Group on Méthodes de détection et de dénombrement de Legionella dans l eau [Methods of detection and enumeration of Legionella in water]. The 2 Ministerial Order of 19 June 2000 amending the Ministerial Order of 14 October 1937 on the control of sources of mineral waters 3 Ministerial Order of 13 December 2004 on general requirements applicable to facilities classified for environmental protection subject to reporting requirements under heading No. 2921: Cooling systems by water dispersion into a flow of air Ministerial Order of 13 December 2004 on Cooling systems by water dispersion into a flow of air, subject to authorisation under heading No Ministerial Order 1 February 2010 on the monitoring of Legionella in hot water production, storage and distribution facilities 2 / 11 ANSES Request No SA-330 methodological and scientific aspects of the work were regularly submitted by the Working Group to the CES. The latter adopted the conclusions on 25 February This expert appraisal was therefore conducted by a group of experts with complementary skills. The scientific aspects of this Opinion are based on the conclusions of the final report from this Expert Committee ( Méthodes de détection et de dénombrement de Legionella dans l eau - Caractéristiques détaillées et pertinence de leur mise en œuvre dans les eaux chaudes sanitaires et les tours aéroréfrigérantes [Methods of detection and enumeration of Legionella in water Detailed characteristics and suitability of their implementation in hot water systems and cooling towers]). 4. DISCUSSION In response to the request, the expert appraisal took account of the following The Legionella species responsible for legionellosis To date, 58 species of bacteria of the genus Legionella have been identified. These species are sub-divided into serogroups (approximately 70 currently known serogroups). In France, Legionella pneumophila is responsible for 98% of reported cases of legionellosis identified from lung samples 5. However, species other than Legionella pneumophila can be the causative agent of legionellosis, especially in patients with weakened immune systems (high-risk patients 6 ). The strains responsible for some of these cases were isolated from hot water supply systems used by or in the vicinity of the patient. In contrast, the literature does not mention any cases of legionellosis linked to a species other than Legionella pneumophila attributed to a cooling tower. In addition, the French data do not include any reported cases of legionellosis attributable to a Legionella species other than pneumophila that may have been caused by cooling tower contamination. However, the difficulty in identifying the environmental source of Legionella cases must be emphasised. In France, where the legionellosis monitoring system is highly developed, its origins still remain unidentified in 80% of cases. Nevertheless, from a health standpoint, Legionella pneumophila appears to be the species most suited to counting, both for hot water and cooling tower water supply systems The health and technical criteria for the choice of the Legionella species for enumeration within the context of regulatory supervision The various regulatory instruments that govern environmental monitoring of Legionella mandate the enumeration of Legionella pneumophila as the most suitable species from a health standpoint, with the exception of the two 13 December 2004 Ministerial Orders on cooling systems by water dispersion into a flow of air, subject to authorisation and notification, commonly referred to as cooling towers. These two Ministerial Orders mandate the enumeration of Legionella spp., even though this choice is not supported from a health standpoint. 5 Respiratory secretions sampling. 66 DGS Circular , of 22 April 2002: high risk patients are defined as severely immunosuppressed, and specifically, immunosuppressed after organ transplants or grafts and those immunosuppressed by prolonged corticosteroid treatment (0.5 mg/kg prednisone for 30 days or more, or equivalent) or recent treatment with a high dose (i.e., more than 5 mg/kg of prednisone for more than 5 days) 3 / 11 ANSES Request No SA-330 Nevertheless, from a technical standpoint, enumerating Legionella spp. may be useful for facilities managers, particularly for detecting malfunctions. However, this issue has not been considered at length in the context of this expert appraisal Criteria to consider when assessing a method for enumeration of Legionella The performance expected from a method for routinely counting microorganisms is assessed on the basis of the following widely accepted criteria: selectivity, specificity, sensitivity, repeatability, reproducibility, validity, reliability, and yield. In the specific context of hot water systems and cooling towers, additional criteria should be taken into account. These criteria were catalogued and prioritised. They include: specific enumeration of Legionella (Legionella spp., Legionella pneumophila, Legionella pneumophila of serogroup 1, other serogroups, etc.); the physiological condition of the Legionella detected; interpretation of the results; the fields of application of the methods (types of water, etc.). A detailed, prioritised list of these criteria is presented in the expert appraisal report and summary corresponding to this Opinion. It is important to take the cost factor into account when choosing an analysis method. However, in this case it must be considered a lower priority than the adequacy of a method for enumerating Legionella in water to ensure effective monitoring of hot water systems and cooling towers. The costs of the particular methods that have been investigated are currently equivalent but are also likely to change in response to future developments. Therefore, this economic aspect has not been dealt with in this expert appraisal and may be assessed once the effectiveness of the techniques their intrinsic characteristics and ability to meet the routine needs of users is properly documented The methods assessed The specific advantages and disadvantages of each method were analysed with regard to the criteria mentioned in Section 4.3. These included: required pretreatment steps: filtration, centrifugation, immunomagnetic separation. specific methods for enumeration of Legionella based on a single principle: o growth: culture, Direct Viable Count (DVC), etc.; o gene amplification: real-time quantitative Polymerase Chain Reaction (q-pcr), viability PCR (v-pcr), etc.; o molecular affinity (immunodetection, fluorescent in situ hybridisation (FISH), etc.; specific methods for counting Legionella based on several principles: immunological double-staining (IDS), culture-fish, and other possible developments (DVC-FISH, matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF), denaturing high pressure liquid chromatography (DHPLC) coupled with PCR, immunomagnetic separation (IMS) measuring Adenosine Triphosphate (ATP-metry); additional analysis techniques: total flora analysis (culture, ATP-metry), detection of amoebae, biofilm analysis, aerosols. A detailed description of these methods is included in the expert appraisal report. From an analysis of the specific advantages and disadvantages of each method, it is clear that the methods that combine the largest number of advantages are culture, q-pcr and, as far as can be determined in the light of the few published examples of its use, v-pcr. However, the process of sorting through this factual information does not cover the respective levels of development of each of the methods, which are often very different, and whether they are suitable for routine application. 4 / 11 ANSES Request No SA Appropriate methods for routine short-term use The choice of an enumeration method cannot be based solely on its intrinsic advantages and disadvantages. Indeed, some advantages identified at the experimental level can fall short when the method confronts the realities in the field. Furthermore, it is important to consider the level of development of the method, i.e., standardisation of the protocol, confirmation of its performance in different laboratories, potential application to different types of environmental samples, robustness, existence of standards and benchmarks, etc. For a routine application within the framework of regulatory supervision, it is important to employ a proven method, whose protocol is validated by multiple laboratories and a large number of samples. Currently, only the methods for enumeration of Legionella in water by culture (French Standards NF T and NF EN ISO ) and by q-pcr (French Standard NF T90-471) offer these assurances, with both of these standards incorporating sample pretreatment. The recommendations for short-term implementation as part of a regulatory framework can therefore reasonably apply only to culture and q-pcr. Other methods seem promising but they need to be developed, stabilised and tested on a large scale, before their routine application can be considered Choice of criteria for interpretation of results according to methods The use of an analytical method also requires criteria defined for interpreting its results. Moreover, before considering the use of an enumeration method for Legionella, within the framework of health monitoring for hot water systems and cooling towers, it is necessary to establish target values above which management actions would be considered. Currently, these target values cannot be based solely on epidemiological data due to: the limited number of publications available on the subject and the considerable uncertainties that they reveal; the difficulty of identifying the environmental source of legionellosis cases (possible in 20% of reported cases); the large number of factors in addition to the concentration of Legionella pneumophila in water from facilities involved in the onset of legionellosis (weather, size of the droplets produced by the facilities, viability status and virulence of the strains of Legionella found in the water, etc.). Also, the determination of target values must be based on a pragmatic approach, by exploiting the available evidence Target values for the culture method In 2001, the French High Council for Public Health (CSHPF) proposed target values based on the scientific knowledge and field observations available at the time. The target values proposed were not based on a dose-response 8 in humans. These target values were incorporated into French regulations. Concerning hot water systems For the general population, the current French regulation sets the L. pneumophila target value at 10 3 colony forming units (CFU)/L. For high-risk (immunocompromised) patients, the regulation advises against exceeding the detection threshold fixed for the culture method (according to French Standard NF T90-431). 7 Given the low number of instances of application of the French Standard NF EN ISO , the current recommendations are primarily based on the Standard NF T Specific relationship between levels of exposure to a hazardous agent and the observed index of a given effect. 5 / 11 ANSES Request No SA-330 According to the data in the literature, the concentration of L. pneumophila below which the legionellosis risk is negligible or acceptable for the general population is between 10 4 or 10 5 CFU/L in hot water systems, despite there being no known dose-response in humans. A review of the literature and interviews with stakeholders involved in the Legionella monitoring system for hot water systems did not produce any evidence to justify amending these target values, from a health standpoint. Concerning cooling towers Current French regulations on cooling towers set the Legionella spp. target values as follows: - acceptability threshold for makeup water: limit of quantification of the standardised method used; - action threshold for installation water: 10 3 CFU/L; - alarm threshold for installation water: 10 5 CFU/L. The literature and consultation with stakeholders in the monitoring system for cooling towers reveal a difficulty in interpreting results related to the choice of a target for counting. Indeed, since the species responsible for most of the reported cases of legionellosis is Legionella pneumophila, it is difficult to construe results exceeding the threshold for Legionella spp. from a health standpoint. These values should primarily be considered as providing information for management purposes Target values for the q-pcr method The current level of knowledge is insufficient for establishing a target value for L. pneumophila with q-pcr (in genome units (GU)/L) based on a dose-response in humans. However, the potential health benefits of using q-pcr, by virtue of its rapid implementation and ability to generate specific results for L. Pneumophila, are incentives for putting tools in place that allow the routine use of this technique. Indeed, in many cases, it would allow detection and thus control of the contamination of an installation much more rapidly than with the conventional culture-based method. In this context, the establishment of target values is paramount. The only current benchmarks are the targets already specified in the regulations for the culture method. The feedback related to their use in monitoring Legionellae in water does not suggest a need to change them. Therefore, it seems reasonable to define the target values applicable to q-pcr so that the proportion of results above these target values, obtained with the same series of samples, would be equivalent for both the culture and q- PCR methods. However, the units used in the different methods for counting Legionella appear to be heterogeneous and not readily comparable, i.e., genome units/l (GU/L) for molecular biology methods and colony forming units/l (CFU/L) for culture methods. Given the lack of published data, the proposed values are primarily based on results from monitoring Legionella in the environment, as indicated by two stakeholders consulted. Concerning hot water systems Based on this reasoning, target values can be developed for potentially hazardous points of use 9 in buildings open to the public, except for those frequented by high-risk patients. 9 Ministerial Order of 1 February 2010 on the monitoring of Legionella in domestic hot water production, storage and distribution facilities, defines potentially hazardous point-of-use as any publicly-accessible point-of-use able to produce aerosols of hot water likely to be contaminated with Legionella; this may include, in particular, showers, showerheads, and jet-whirlpool baths. 6 / 1
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